Vitamin D3 is metabolized to 25-hydroxy-vitamin D3 (25-OH-D3) in several tissues and the 25-OH-D3 metabolite is further converted by the kidney to 1,25-dihydroxy-vitamin D3 (1,25-(OH)2-D3). This latter sterol appears to be the hormonal form of the vitamin which carries out the biochemical functions heretofore ascribed to vitamin D. The vitamin D-25-hydroxylase of liver and kidney and the 25- hydroxy-vitamin D3-1-hydroxylase (kidney) will be studied at four levels: 1) whole animals, 2) cells grown in culture, 3) cellular homogenates, and 4) purified enzymes. Substrate specificity, kinetic characteristics, cofactor requirements, and possible regulatory effects of physiologic and pharmacologic factors will be investigated for these two enzymes. Special attention will be paid to parathyroid hormone as a possible controlling agent for the production of 1,25-(OH)2-D3. Another objective will be the elucidation of the molecular mode of action of 1,25-(OH)2-D3 in intestine and bone. Rachitic chicks, as well as intestine and bone grown in organ culture will be employed for direct experiments with 1,25-(OH)2-D3. Time course of binding to target cells and the nature of receptor molecules for 1,25-(OH)2-D3 in the intestine will be studied. The nuclear receptor molecule for 1,25-(OH)2-D3 in the intestine will be isolated via chromatography and (in combination with hormone) tested for biochemical efficacy in isolated systems such as DNA transcription. In addition, investigation of the effects of 1,25-(OH)2- D3 treatment on RNA synthesis will be performed using labeled RNA precursors and disc gel electrophoresis to fractionate the RNA.